Crystal structures of MK2 and uses thereof

ABSTRACT

This invention is directed to the crystal structures of MK2, and to the use of these structures in rational drug design methods to identify agents that may interact with active sites of MK2 proteins. Such agents may be useful as therapeutic agents.

RELATED APPLICATION

This application is a continuation application of U.S. patent application Ser. No. 10/294,027, filed Nov. 13, 2002. The contents of the prior application is hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to the identification of the crystal structures of MK2, and the use of the structures for designing new drugs.

BACKGROUND OF THE INVENTION

Mitogen activated protein (MAP) kinases are a large and diverse group of Ser/Thr kinases separated into three major subgroups, which include the extracellular signal regulated kinases (ERKs), the c-Jun N-terminal kinases (JNKs)/stress-activated protein kinases (JNKs) and p38/reactivating kinases (RK). The ERKs are activated by mitogens and growth factors, whereas the JNKs/SAPKs and p38/RK are activated by bacterial lipopolysaccharide (LPS, interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α) and cellular stresses such as heat shock, osmotic shock, or UV damage. Exposure of cells to these factors results in the increased production of proinflammatory cytokines. Analysis of a specific inhibitor of p38 MAP kinase, SB203580, reveals that it inhibits LPS-induced cytokine synthesis in human monocytes, thus indicating that p38 is the MAP kinase responsible for stress-induced cytokine production (1). SB203580 also prevents the activation of MAP kinase activated protein kinase 2 (MK2), suggesting that this kinase is activated by P38 (2).

Mice engineered to be homozygously-deficient in MK2 show a reduction in TNF-α, interferon-γ, IL-1β, and IL-6 production and an increased rate of survival upon challenge with LPS, suggesting that this enzyme is a key component in the inflammatory process and a potential target for anti-inflammatory therapy (3). Activation of MK2 results in the production of cytokines by regulating the translation and or stability of the encoding mRNAs through the AU-rich elements of the 3′-untranslated regions of the gene (4). MK2 also phosphorylates the transcription factor CREB, as well as leukocyte specific protein-1 and heat shock protein 25/27, which are involved in the regulation of actin polymerization (5-8) and cell migration (9, 10).

MK2 is a multi-domain protein consisting of an N-terminal proline-rich domain, a catalytic domain, an autoinhibitory domain and at the C-terminus a nuclear export signal (NES) and nuclear localization signal (NLS) (11-15). Two isoforms of human MK2 have been characterized. One isoform consists of 400 amino acids and the other isoform 370 residues which is thought to be a splice variant missing the C-terminal NLS (11, 16, 12). MK2 is located in the nucleus of the cell and upon binding and phosphorylation by p38, the MK2 NES becomes functional and both kinases are co-transported out of the nucleus to the cytoplasm (8, 12, 13, 17). Interestingly, transport of the MK2/p38 complex does not require catalytically active MK2, as the active site mutant, Asp207Ala, is still transported to the cytoplasm (13). Phosphorylation of human MK2 by p38 on residues T222, S272 and T334 is thought to activate the enzyme by inducing a conformational change of the autoinhibitory domain thus exposing the active site for substrate binding (8, 18). Mutations of two autoinhibitory domain residues W332A and K326E in murine MK2 demonstrate an increase in basal activity and a C-terminal deletion of the autoinhibitory domain renders the enzyme constitutively active, providing additional evidence to the role of this domain in inhibition of MK2 activity (18).

Recently, Meng, et al., published the structure of the autoinhibited, inactive form of MK2 47-400. However, since MK2 47-400 used by Meng, et al. included the autoinhibitory domain and was otherwise inactive, that structure is less useful for drug design.

SUMMARY OF THE INVENTION

The present invention provides an isolated MK2 polypeptide, having an amino acid sequence corresponding to a portion of MK2, in which the N-terminus begins at amino acid 41 to 55 and the C-terminus ends at 338 to 365, or an MK2 analogue thereof. This MK2 polypeptide, unlike the MK2 polypeptide used by Meng, et al., does not include the complete autoinhibitory domain and is catalytically active.

The present invention also provides nucleic acids encoding the forgoing MK2 polypeptides or MK2 analogues thereof, vectors comprising the nucleic acids, as well as host cells transformed, transfected or infected with the vectors. Additionally, the present invention provides a method for preparing an MK2 polypeptide or an MK2 analogue that comprises transforming, transfecting or infecting a host cell with the vector, and culturing the host cell under conditions permitting the production of MK2 polypeptide or MK2 analogue by the host cell.

The present invention also provides a method for obtaining crystallized native MK2, an MK2 polypeptide or an MK2 analogue comprising contacting native MK2, an MK2 polypeptide or an MK2 analogue with a buffer solution comprising at least one of cacodylate, Tris, Tris-HCL, acetate, malonate, sodium phosphate, potassium phosphate, citrate, HEPES and MES, at a salt concentration of 0.1 M to 2.4 M, and at a pH of 4.5 to 8.5, under conditions permitting the formation of crystallized MK2, crystallized MK2 polypeptide or crystallized MK2 analogue.

The present invention also provides a crystallized complex of MK2 polypeptide and staurosporine, having four molecules of MK2 in the asymmetric unit.

Additionally, the present invention provides a crystallized complex of MK2 polypeptide and ADP, having one molecule of MK2 in the asymmetric unit.

The present invention further provides a three dimensional model of MK2, defined by the relative structural coordinates for: (i) molecules A, B, C or D of MK2 according to FIGS. 2 through 2A-204, (ii) a portion of molecules A, B, C, or D of MK2 according to FIGS. 2 through 2A-204, (iii) molecule A of MK2 according to FIGS. 3 through 3A-40, or (iv) a portion of molecule A of MK2 according to FIGS. 3 through 3A-40, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å.

Also provided by the present invention is an active site of MK2, and particularly the site on MK2 in which staurosporine binds. The active site comprises the relative structural coordinates of amino acid resides Leu70, Gly71, Leu72, Gly73, Val78, Ala91, Val118, Mse138, Glu139, Cys140, Leu141, Glu145, Glu190, Asn191, Leu192, Thr206 and Asp207 of molecules A, B, C or D according to FIGS. 2 through 2A-204, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å. In another embodiment, the active site further comprises, in addition to the above relative coordinates, the relative structural coordinates for amino acid residues Val69, Ile74, Gly76, Ala77, Leu79, Gln80, Lys89, Phe90, Leu92, Lys93, Leu95, Glu104, His108, Arg119, Ile136, Val137, Asp142, Gly143, Gly144, His184, Asp186, Lys188, Pro189, Leu193, Tyr194, Thr195, Lys204, Leu205, Phe208 and Gly209 of molecules A, B, C or D according to FIGS. 2 through 2A-204, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å.

The present invention also provides a method for designing a putative agent that interacts with an active site of MK2, and particularly the staurosporine binding site on MK2. This method comprises the steps of: (a) generating a three dimensional model of said active site using the relative structural coordinates of amino acid residues Leu70, Gly71, Leu72, Gly73, Val78, Ala91, Val118, Mse138, Glu139, Cys140, Leu141, Glu145, Glu190, Asn191, Leu192, Thr206, and Asp207 of molecules A, B, C or D of MK2 according to FIGS. 2 through 2A-204, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 521; and (b) designing a putative agent that interacts with said active site by performing computer fitting analysis of said putative agent with the three dimensional model generated in step (a). In another embodiment, the active site further comprises, in addition to the above relative coordinates, the relative structural coordinates for amino acid residues Val69, Ile74, Gly76, Ala77, Leu79, Gln80, Lys89, Phe90, Leu92, Lys93, Leu95, Glu104, His108, Arg119, Ile136, Val137, Asp142, Gly143, Gly144, His184, Asp186, Lys188, Pro189, Leu193, Tyr194, Thr195, Lys204, Leu205, Phe208 and Gly209 of molecules A, B, C or D according to FIGS. 2 through 2A-204, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å.

Still further, the present invention provides a method for identifying an agent that interacts with MK2, comprising the steps of: (a) generating a three dimensional model of MK2 using the relative structural coordinates of (i) molecules A, B, C or D of MK2 according to FIGS. 2 through 2A-204, (ii) a portion of molecules A, B, C or D of MK2 according to FIGS. 2 through 2A-204, (iii) molecule A of MK2 according to FIGS. 3 through 3A-40, or (iv) a portion of molecule A of MK2 according to FIGS. 3 through 3A-40, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å; and (b) employing said three-dimensional model to identify an agent that interacts with MK2.

The present invention also provides a method for designing a putative agent that interacts with an active site of MK2, and particularly the staurosporine binding site on MK2. This method comprises the steps of: (a) generating a three dimensional model of said active site using the relative structural coordinates of amino acid residues Leu70, Gly71, Leu72, Gly73, Val78, Ala91, Val118, Mse138, Glu139, Cys140, Leu141, Glu145, Glu190, Asn191, Leu192, Thr206, and Asp207 of molecules A, B, C or D of MK2 according to FIGS. 2 through 2A-204, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å; and (b) designing a putative agent that interacts with said active site by performing computer fitting analysis of said putative agent with the three dimensional model generated in step (a). In another embodiment, the active site further comprises, in addition to the above relative coordinates, the relative structural coordinates for amino acid residues Val69, Ile74, Gly76, Ala77, Leu79, Gln80, Lys89, Phe90, Leu92, Lys93, Leu95, Glu104, His108, Arg119, Ile136, Val137, Asp142, Gly143, Gly144, His184, Asp186, Lys188, Pro189, Leu193, Tyr194, Thr195, Lys204, Leu205, Phe208 and Gly209 of molecules A, B, C or D according to FIGS. 2 through 2A-204, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å.

Additionally, the present invention provides a method for identifying a putative agent that interacts with an active site of MK2, and particularly the site on MK2 in which ADP binds. This method comprises the steps of: (a) generating a three dimensional model of said active site using the relative structural coordinates of amino acid residues Leu70, Gly71, Leu72, Gly73, Ile74, Asn75, Val78, Ala91, Lys93, Met138, Glu139, Cys140, Leu141, Asn191, Thr206, Asp207 of molecule A of MK2 according to FIGS. 3 through 3A-40, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å; and (b) designing a putative agent that interacts with said active site by performing computer fitting analysis of said putative agent with the three dimensional model generated in step (a). In another embodiment, the active site further comprises, in addition to the above relative coordinates, the relative structural coordinates for amino acid residues Val69, Gly76, Ala77, Leu79, Gln80, Phe90, Leu92, Met94, Leu95, Glu104, His108, Val118, Ala119, Ile136, Val137, Asp142, Gly143, Gly144, Glu145, His184, Asp186, Lys188, Pro189, Glu190, Leu192, Leu193, Tyr194, Thr195, Leu205, Phe208, Gly209, Phe210 of molecule A of MK2 according to FIGS. 3 through 3A-40, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å.

Finally, the present invention provides agents identified using the foregoing methods. Small molecules or other agents which activate, inhibit or otherwise interfere with substrate binding to MK2 may be useful as therapeutic agents in inflammatory based diseases.

Additional objects of the present invention will be apparent from the description which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a sequence alignment of MK2 (SEQ ID NO:1), MK3 (SEQ ID NO:2) and MK5 (SEQ ID NO:3). Residues highlighted in bold are identical. The GXGXXG motif (SEQ ID NO:6), the catalytic RD residues and the bipartite nuclear localization sequence (KKI . . . RKK) are boxed. The residues that are phosphorylated are donated by stars.

FIGS. 2 through 2A-204 provide the atomic structural coordinates for MK2 and staurosporine as derived by X-ray diffraction of the MK2/staurosporine crystal complex. “Atom type” refers to the atom whose coordinates are being measured. “Residue” refers to the type of residue of which each measured atom is a part—i.e., amino acid, cofactor, ligand or solvent. The “x, y and z” coordinates indicate the Cartesian coordinates of each measured atom's location in the unit cell (Å). “Occ” indicates the occupancy factor. “B” indicates the “B-value”, which is a measure of how mobile the atom is in the atomic structure (Å²). Under “Molecules”, A, B, C, and D refer to each molecule of MK2, E, F G and H refer to each molecule of staurosporine, molecule I corresponds to SO₄, and W corresponds to water.

FIGS. 3 through 3A-40 provide the atomic structural coordinates for MK2 and ADP as derived by X-ray diffraction of the MK2/ADP crystal complex. “Atom type” refers to the atom whose coordinates are being measured. “Residue” refers to the type of residue of which each measured atom is a part—i.e., amino acid, cofactor, ligand or solvent. The “x, y and z” coordinates indicate the Cartesian coordinates of each measured atom's location in the unit cell (Å). “Occ” indicates the occupancy factor. “B” indicates the “B-value”, which is a measure of how mobile the atom is in the atomic structure (Å²) Under “Molecule”, A refers to MK2 and B corresponds to ADP.

FIGS. 4 through 4A-1 provide the nucleic acid sequence for MK2 (SEQ ID NO:5) with the corresponding MK2 amino acid sequence (SEQ ID NO:4).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following terms and phrases shall have the meanings set forth below:

Unless otherwise noted, “MK2” includes: (i) native MK2 having the amino acid sequence (residues 1-400) set forth in FIG. 1, including conservative substitutions thereof; (ii) modeled MK2, including conservative substitutions thereof; and (iii) an MK2 polypeptide, including conservative substitutions thereof.

“Modeled MK2” corresponds to molecules A, B, C and D of MK2 according to FIGS. 2 through 2A-204, and molecule A of MK2 according to FIGS. 3 through 3A-40.

An “MK2 polypeptide” is an amino acid sequence that defines a portion or fragment of residues 1-400 of MK2 set forth in FIG. 1. An MK2 polypeptide includes but is not limited to an amino acid sequence of MK2 in which the N-terminus begins at amino acid 41 to 55 and the C-terminus ends at 338 to 365, in accordance with the residue numbering shown in FIG. 1.

An “MK2 analogue, is a polypeptide having at least 80% homology with “MK2” defined above, more preferably at least 90% homology with “MK2” defined above, and most preferably at least 95% homology with “MK2” defined above. In the preferred embodiment, an “MK2 analogue” also has MAP kinase activated protein kinase activity.

A “portion” of molecules A, B, C, or D of MK2 set forth in FIGS. 2 through 2A-204, or a “portion” of molecule A of MK2 set forth in FIGS. 3 through 3A-40, refers to the relative structural coordinates of amino acid residues that include less than all the amino acid residues shown. Preferably, the portion of molecules A, B, C or D of MK2 set forth in FIGS. 2 through 2A-204, or a portion of molecule A of MK2 set forth in FIGS. 3 through 3A-40 includes, at a minimum, a sufficient portion of the MK2 molecule to define an active site of MK2.

An “MK2 complex” is MK2 complexed to another molecule, including but not limited to staurosporine or ADP.

Unless otherwise indicated, “protein” or “molecule” shall include a protein, protein domain, polypeptide or peptide.

“Structural coordinates” are the Cartesian coordinates corresponding to an atom's spatial relationship to other atoms in a molecule or molecular complex. Structural coordinates may be obtained using x-ray crystallography techniques or NMR techniques, or may be derived using molecular replacement analysis or homology modeling. Various software programs allow for the graphical representation of a set of structural coordinates to obtain a three dimensional representation of a molecule or molecular complex. The structural coordinates of the present invention may be modified from the original sets provided in FIGS. 2 through 2A-204 and FIGS. 3 through 3A-40 by mathematical manipulation, such as by inversion or integer additions or subtractions. As such, it is recognized that the structural coordinates of the present invention are relative, and are in no way specifically limited by the actual x, y, z coordinates of FIGS. 2 through 2A-204 and FIGS. 3 through 3A-40.

An “agent” shall include a protein, polypeptide, peptide, nucleic acid (including DNA or RNA), molecule, compound or drug.

“Root mean square deviation” is the square root of the arithmetic mean of the squares of the deviations from the mean, and is a way of expressing deviation or variation from the structural coordinates described herein. The present invention includes all embodiments comprising conservative substitutions of the noted amino acid residues resulting in same structural coordinates within the stated root mean square deviation. It will be obvious to the skilled practitioner that the numbering of the amino acid residues of MK2 may be different than that set forth herein, and may contain certain conservative amino acid substitutions that yield the same three dimensional structures as those defined by FIGS. 2 through 2A-204 and FIGS. 3 through 3A-40 herein. Corresponding amino acids and conservative substitutions in other isoforms or analogues are easily identified by visual inspection of the relevant amino acid sequences or by using commercially available homology software programs (e.g., MODELLAR, MSI, San Diego, Calif.).

“Conservative substitutions” are those amino acid substitutions which are functionally equivalent to the substituted amino acid residue, either by way of having similar polarity, steric arrangement, or by belonging to the same class as the substituted residue (e.g., hydrophobic, acidic or basic), and includes substitutions having an inconsequential effect on the three dimensional structure of MK2 with respect to the use of said structures for the identification and design of agents which interact with MK2, as well as for molecular replacement analyses and/or for homology modeling.

The present invention first provides an MK2 polypeptide having an amino acid sequence corresponding to a portion of MK2, in which the N-terminus begins at amino acid 41 to 55 and the C-terminus ends at 338 to 365. The present inventors have found that sequences which lack amino acid residues 366-400 of MK2, are active. Preferably, a MK2 polypeptide has the amino acid sequence corresponding to amino acid residues 41-364 of MK2 according to FIG. 1.

The present invention also provides an MK2 analogue of the foregoing MK2 polypeptide that is at least 80% homologous, more preferably at least 90% homologous and most preferably at least 95% homologous, with the foregoing MK2 polypeptide having an amino acid sequence corresponding to a portion of MK2, in which the N-terminus begins at amino acid 41-to 55 and the C-terminus ends at 338 to 365. In the more preferred embodiment, the MK2 analogue is at least 80% homologous, more preferably at least 90% homologous and most preferably at least 95% homologous, with the MK2 polypeptide having the amino acid sequence corresponding to amino acid residues 41-364 of MK2 according to FIG. 1.

The present invention also provides a nucleic acid encoding the MK2 polypeptide having an amino acid sequence corresponding to a portion of MK2, in which the N-terminus begins at amino acid 41 to 55 and the C-terminus ends at 338 to 365, as well as a nucleic acid encoding amino acid residues 41-364 of MK2 according to FIG. 1. The nucleic acid sequence for MK2 is known, and is shown in FIGS. 4 through 4A-1. Such nucleic acids can be introduced into the appropriate vectors, and then transformed, transfected or infected into the appropriate host cells, which can be cultured to produce recombinant MK2 polypeptide. The recombinant MK2 polypeptide can then be isolated and purified according to known techniques. Suitable vector and expression systems are well known and commercially available.

The present invention also provides a nucleic acid encoding an MK2 analogue that is at least 80% homologous, more preferably at least 90% homologous and most preferably at least 95% homologous, with the MK2 polypeptide having an amino acid sequence corresponding to a portion of MK2, in which the N-terminus begins at amino acid 41 to 55 and the C-terminus ends at 338 to 365. In the more preferred embodiment, the nucleic acid encodes an MK2 analogue that is at least 80% homologous, more preferably at least 90% homologous and most preferably at least 95% homologous, with the MK2 polypeptide having the amino acid sequence corresponding to amino acid residues 41-364 of MK2 according to FIG. 1. Such nucleic acids can be introduced into the appropriate vectors, and then transformed, transfected or infected into the appropriate host cells, which can be cultured to produce recombinant MK2 polypeptide. The recombinant MK2 analogue can then be isolated and purified according to known techniques. Suitable vector and expression systems are well known and commercially available.

The present invention also provides a method for crystallizing native MK2, an MK2 polypeptide or an MK2 analogue. The method comprises contacting native MK2, an MK2 polypeptide or an MK2 analogue with a buffer solution comprising at least one of cacodylate, Tris, Tris-HCL, acetate, malonate, sodium phosphate, potassium phosphate, citrate, HEPES and MES, at a salt concentration of 0.1 M to 2.4 M, and at a pH of 4.5 to 8.5, under conditions permitting the formation of crystallized native MK2, crystallized MK2 polypeptide or crystallized MK2 analogue. In a preferred embodiment, the salt has an anion selected from the group consisting of sulfate, citrate, chloride, acetate, phosphate, malonate and tartrate. In another preferred embodiment, the salt concentration is 0.8 M or higher. It is also within the confines of the present invention that the native MK2, the MK2 polypeptide or the MK2 analogue is contacted with the buffer solution in the presence of PEG or a PEG substitute having a molecular weight up to 3350. The PEG includes but is not limited to PEG-200, PEG-400, PEG-500-MME, PEG-1000, PEG-1500, PEG-2000-MME and MEG-3350, and is preferably PEG-400. The PEG substitute includes but is not limited to Jeffamine M-600, ethylene glycol, glycerol and 1-6 hexanediol, 2-methyl-2,4-pentanediol (MPD). The more specific crystallization conditions are exemplified in example which follows, as well as in Table 4.

The present invention also provides a crystallized complex of MK2 polypeptide and staurosporine, having four molecules of MK2 polypeptide in the asymmetric unit. This crystal effectively diffracts X-rays for the determination of the structural coordinates of MK2, and is characterized as having space group P6₃, unit cell parameters of a=b=160.20 Å, c=133.48 Å.

Additionally, the present invention provides a crystallized complex of MK2 polypeptide and ADP, having one molecule of MK2 polypeptide in the asymmetric unit. This crystal effectively diffracts X-rays for the determination of the structural coordinates of MK2, and is characterized as having space group F4₁32, unit cell parameters of a=b=c=253.05 Å.

Using the crystals of the present invention, X-ray diffraction data can be collected by a variety of means in order to obtain the atomic coordinates of the molecules in the crystals. With the aid of specifically designed computer software, such crystallographic data can be used to generate a three dimensional structure. Various methods used to generate and refine a three dimensional structure of a molecular structure are well known to those skilled in the art, and include, without limitation, multiwavelength anomalous dispersion (MAD), multiple isomorphous replacement, reciprocal space solvent flattening, molecular replacement, and single isomorphous replacement with anomalous scattering (SIRAS).

Accordingly, the present invention also provides a three dimensional model of MK2 as derived by x-ray diffraction data of the MK2/staurosporine crystal. The three dimensional model of MK2 derived from the MK2/staurosporine crystal is preferably defined by the relative structural coordinates for molecules A, B, C or D of MK2 according to FIGS. 2 through 2A-204, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å, preferably not more than 1.0 Å, and most preferably not more than 0.5 Å. The three dimensional model also includes the relative structural coordinates of a portion of molecules A, B, C or D of MK2 according to FIGS. 2 through 2A-204, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å, preferably not more than 1.0 Å, and most preferably not more than 0.5 Å. The three dimensional model of MK2 is useful for a number of applications, including, but not limited to, the visualization, identification and characterization of various active sites of MK2. The active site structures may then be used to design agents with interact with MK2.

The present invention also provides a three dimensional model of MK2 as derived by x-ray diffraction data of the MK2/ADP crystal. The three dimensional model of MK2 derived from the MK2/ADP crystal is preferably defined by the structural coordinates for molecule A of MK2 shown in FIGS. 3 through 3A-40, ±a root mean square deviation from the backbone atoms of the amino acids of not more than 1.5 Å, preferably not more than 1.0 Å, and most preferably not more than 0.5 Å. The three dimensional model also includes the relative structural coordinates of a portion of molecule A of MK2 according to FIGS. 3 through 3A-40, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å, preferably not more than 1.0 Å, and most preferably not more than 0.5 Å. The three dimensional model of MK2 derived from the MK2/ADP crystal is useful for a number of applications, including, but not limited to, the visualization, identification and characterization of various active sites of MK2. The active site structures may then be used to design agents with interact with MK2.

The present invention also provides a machine, such as a computer, programmed in memory with the coordinates of FIGS. 2 through 2A-204, or FIGS. 3 through 3A-40, or portions thereof, together with a program capable of converting the coordinates into a three dimensional graphical representation of the structural coordinates on a display connected to the machine. A machine having a memory containing such data aids in the rational design or selection of inhibitors or activators of MK2 activity, including the evaluation of ability of a particular chemical entity to favorably associate with MK2 or an MK2 complex as disclosed herein, as well as in the modeling of compounds, proteins, complexes, etc. related by structural or sequence homology to MK2.

For storage, transfer and use with such programs, a machine, such as a computer, is provided for that produces a three dimensional representation of the MK2 molecule, a portion thereof (such as an active site or a binding site), an MK2 molecular complex, or an MK2 analogue. The machine of the present invention comprises a machine-readable data storage medium comprising a data storage material encoded with machine-readable data. Machine-readable storage media comprising data storage material include conventional computer hard drives, floppy disks, DAT tape, CD-ROM, and other magnetic, magneto-optical, optical, and other media which may be adapted for use with a computer. The machine of the present invention also comprises a working memory for storing instructions for processing the machine-readable data, as well as a central processing unit (CPU) coupled to the working memory and to the machine-readable data storage medium for the purpose of processing the machine-readable data into the desired three dimensional representation. Finally, the machine of the present invention further comprises a display connected to the CPU so that the three dimensional representation may be visualized by the user. Accordingly, when used with a machine programmed with instructions for using said data, e.g., a computer loaded with one or more programs of the sort identified below, the machine provided for herein is capable of displaying a graphical three-dimensional representation of any of the molecules or molecular complexes, or portions of molecules of molecular complexes, described herein.

Molecular modeling methods known in the art may be used to identify an active site or binding pocket of MK2, MK2 complex or an MK2 analogue. Specifically, the structural coordinates provided by the present invention may be used to characterize a three dimensional model of the MK2, MK2 complex or MK2 analogue. From such a model, putative active sites may be computationally visualized, identified and characterized based on the surface structure of the molecule, surface charge, steric arrangement, the presence of reactive amino acids, regions of hydrophobicity or hydrophilicity, etc. Such putative active sites may be further refined using chemical shift perturbations of spectra generated from various and distinct MK2 complexes, competitive and non-competitive inhibition experiments, and/or by the generation and characterization of MK2 mutants to identify critical residues or characteristics of the active site. The identification of putative active sites of a molecule or molecular complex is of great importance, as most often the biological activity of a molecule or molecular complex results from the interaction between an agent and one or more active sites of the molecule or molecular complex. Accordingly, the active sites of a molecule or molecular complex are the best targets to use in the design or selection of activators or inhibitors that affect the activity of the molecule or molecular complex.

As such, the present invention also provides an active site of MK2, and particularly the site of binding of staurosporine to MK2. In one embodiment, the active site comprises the relative structural coordinates of amino acid residues Leu70, Gly71, Leu72, Gly73, Val78, Ala91, Val118, Mse138, Glu139, Cys140, Leu141, Glu145, Glu190, Asn191, Leu192, Thr206 and Asp207 of molecules A, B, C or D according to FIGS. 2 through 2A-204, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å, preferably not more than 1.0 Å, and most preferably not more than 0.5 ÅIn another embodiment, the active site further comprises, in addition to the above relative coordinates, the relative structural coordinates for amino acid residues Val69, Ile74, Gly76, Ala77, Leu79, Gln80, Lys89, Phe90, Leu92, Lys93, Leu95, Glu104, His108, Arg119, Ile136, Val137, Asp142, Gly143, Gly144, His184, Asp186, Lys188, Pro189, Leu193, Tyr194, Thr195, Lys204, Leu205, Phe208 and Gly209 of molecules A, B, C or D according to FIGS. 2 through 2A-204, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å, preferably not more than 1.0 Å, and most preferably not more than 0.5 Å.

Still further, the present invention provides an active site of MK2, and particularly the site of binding of staurosporine to ADP. In one embodiment, the active site comprises the relative structural coordinates of amino acid residues Leu70, Gly71, Leu72, Gly73, Ile74, Asn75, Val78, Ala91, Lys93, Met138, Glu139, Cys140, Leu141, Asn191, Thr206, Asp207 of molecule A of MK2 according to FIGS. 3 through 3A-40, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å, preferably not more than 1.0 Å, and most preferably not more than 0.5 Å. In another embodiment, the active site further comprises, in addition to the above relative coordinates, the relative structural coordinates for amino acid residues Val69, Gly76, Ala77, Leu79, Gln80, Phe90, Leu92, Met94, Leu95, Glu104, His108, Val118, Ala119, Ile136, Val137, Asp142, Gly143, Gly144, Glu145, His184, Asp186, Lys188, Pro189, Glu190, Leu192, Leu193, Tyr194, Thr195, Leu205, Phe208, Gly209, Phe210 of molecule A of MK2 according to FIGS. 3 through 3A-40, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å, preferably not more than 1.0 Å, and most preferably not more than 0.5 Å.

Another aspect of the present invention is directed to a method for identifying an agent that interacts with MK2. In this method, a three dimensional model of MK2 is first generated using the relative structural coordinates of (i) molecules A, B, C or D of MK2 according to FIGS. 2 through 2A-204, (ii) a portion of molecules A, B, C or D of MK2 according to FIGS. 2 through 2A-204, (iii) molecule A of MK2 according to FIGS. 3 through 3A-40, or (iv) a portion of molecule A according to FIGS. 3 through 3A-40, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å, preferably not more than 1.0 Å, and most preferably not more than 0.5 Å. The three-dimensional model is then used to identify an agent that interacts with MK2.

The present invention also provides a method for designing a putative agent that interacts with an active site of MK2, and particularly the site on MK2 to which staurosporine binds. In this method, a three dimensional model of the active site is first generated using the relative structural coordinates of amino acid residues Leu70, Gly71, Leu72, Gly73, Val78, Ala91, Val118, Mse138, Glu139, Cys140, Leu141, Glu145, Glu190, Asn191, Leu192, Thr206, and Asp207 of molecules A, B, C or D of MK2 according to FIGS. 2 through 2A-204, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å, preferably not more than 1.0 Å, and most preferably not more than 0.5 Å. A putative agent that interacts with the active site is then designed, generated or identified by performing computer fitting analysis of the putative agent with the three dimensional model generated above. In another embodiment, the active site further comprises, in addition to the above relative coordinates, the relative structural coordinates for amino acid residues Val69, Ile74, Gly76, Ala77, Leu79, Gln80, Lys89, Phe90, Leu92, Lys93, Leu95, Glu104, His108, Arg119, Ile136, Val137, Asp142, Gly143, Gly144, His184, Asp186, Lys188, Pro189, Leu193, Tyr194, Thr195, Lys204, Leu205, Phe208 and Gly209 of molecules A, B, C or D according to FIGS. 2 through 2A-204, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å, preferably not more than 1.0 Å, and most preferably not more than 0.5 Å.

The present invention also provides a method for designing a putative agent that interacts with an active site of MK2, and particularly the site on MK2 to which staurosporine binds. In this method, a three dimensional model of the active site is first generated using the relative structural coordinates of amino acid residues Leu70, Gly71, Leu72, Gly73, Val78, Ala9, Val118, Mse138, Glu139, Cys140, Leu141, Glu145, Glu190, Asn191, Leu192, Thr206, and Asp207 of molecules A, B, C or D of MK2 according to FIGS. 2 through 2A-204, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å, preferably not more than 1.0 Å, and most preferably not more than 0.5 Å. A putative agent that interacts with the active site is then designed, generated or identified by performing computer fitting analysis of the putative agent with the three dimensional model generated above. In another embodiment, the active site further comprises, in addition to the above relative coordinates, the relative structural coordinates for amino acid residues Val69, Ile74, Gly76, Ala77, Leu79, Gln80, Lys89, Phe90, Leu92, Lys93, Leu95, Glu104, His108, Arg119, Ile136, Val137, Asp142, Gly143, Gly144, His184, Asp186, Lys188, Pro189, Leu193, Tyr194, Thr195, Lys204, Leu205, Phe208 and Gly209 of molecules A, B, C or D according to FIGS. 2 through 2A-204, ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å, preferably not more than 1.0 Å, and most preferably not more than 0.5 Å.

Using the active site, the agent may be designed or evaluated using computer fitting analyses utilizing various computer software programs that evaluate the “fit” between the putative active site and the identified agent, by (a) generating a three dimensional model of the putative active site of a molecule or molecular complex using homology modeling or the atomic structural coordinates of the active site, and (b) determining the degree of association between the putative active site and the identified agent. The degree of association may be determined computationally by any number of commercially available software programs, or may be determined experimentally using standard binding assays.

Three dimensional models of the putative active site may be generated using any one of a number of methods known in the art, and include, but are not limited to, homology modeling as well as computer analysis of raw data generated using crystallographic or spectroscopy data. Computer programs used to generate such three dimensional models and/or perform the necessary fitting analyses include, but are not limited to: GRID (Oxford University, Oxford, UK), MCSS (Molecular Simulations, San Diego, Calif.), AUTODOCK (Scripps Research Institute, La Jolla, Calif.), DOCK (University of California, San Francisco, Calif.), Flo99 (Thistlesoft, Morris Township, N.J.), Ludi (Molecular Simulations, San Diego, Calif.), QUANTA (Molecular Simulations, San Diego, Calif.), Insight (Molecular Simulations, San Diego, Calif.), SYBYL (TRIPOS, Inc., St. Louis, Mo.) and LEAPFROG (TRIPOS, Inc., St. Louis, Mo.). The structural coordinates also may be used to visualize the three-dimensional structure of MK2 using MOLSCRIPT (Kraulis, P J, J. Appl. Crystallogr. 24: 946-950 (1991)) and RASTER3D (Bacon, D. J. and Anderson, W. F., J. Mol. Graph. 6: 219-220 (1998)), for example.

The agent, whether an inhibitor or activator, may be selected by screening an appropriate database, may designed de novo by analyzing the steric configurations and charge potentials of an empty MK2 active site in conjunction with the appropriate software programs, or may be designed using characteristics of known inhibitors or activators to MK2 or other mitogen activated protein kinases in order to create “hybrid” activators or inhibitors. The method of the present invention is preferably used to design or select inhibitors of MK2. In this case, the potential inhibitor or activator is designed to incorporate chemical or steric features favorable for association with the active site. The inhibitor or activator may be selected by screening an appropriate database, may designed de novo by analyzing the steric configurations and charge potentials of empty active sites in conjunction with the appropriate software programs, or may be designed using characteristics of known inhibitors or activators to MK2 or other mitogen activated protein kinases in order to create “hybrid” activators or inhibitors.

Once the agent has been designed or identified, it may be obtained or synthesized and further evaluated for its affect on MK2 activity. For example, the agent may be evaluated by contacting the identified agent with MK2 and measuring the effect of the agent on MK2 activity. Depending upon the action of the agent on MK2, the agent may act either as an inhibitor or activator of MK2 activity. With respect to the specific active sites identified above, the agent also may be contacted with MK2 in the presence of staurosporine or ADP in order to determine whether or not the agent inhibits binding between MK2 and staurosporine or ADP, respectively.

Various molecular analysis and rational drug design techniques are further disclosed in U.S. Pat. Nos. 5,834,228, 5,939,528 and 5,865,116, as well as in PCT Application No. PCT/US98/16879, published WO 99/09148, the contents of which are hereby incorporated by reference.

The present invention is also directed to the agents, activators or inhibitors identified using the foregoing methods. Such agents may be a protein, polypeptide, peptide, nucleic acid, including DNA or RNA, molecule, compound, or drug. Small molecules or other agents which interact with MK2 may be useful in the treatment of diseases or conditions associated with MK2.

The present invention may be better understood by reference to the following non-limiting Example. The following Example is presented in order to more fully illustrate the preferred embodiments of the invention, and should in no way be construed as limiting the scope of the present invention.

EXAMPLE 1

1. Materials and Methods.

Sequence Selection for MK2 Crystallization. Various constructs were made, expressed and the resulting MK2 were purified and crystalized. After analyzing a large number of constructs for expression of MK2 in E. coli, it was found that deleting the first 40 residues of the proline rich domain greatly enhanced the expression levels and solubility of the enzyme. Analyses of the resulting MK2 also indicated that constructs lacking amino acids 366 to 400 were not active in vitro. It was also found that crystals could be obtained for constructs of MK2 in which the N-terminus begins at amino acid 41 to 55 and the C-terminus ends at 338 to 365. The construct 41-364 resulted in the best crystals for determining the crystal structure of MK2, and details concerning its expression, purification and crystallization are provided below.

Cloning and expression of MK2. The MAPKAP kinase 2 gene was PCR cloned into the NcoI and XhoI sites of pET16b (Novagen) using Hot tub polymerase (Amersham Pharmacia Biotech). The expressed protein contains residues 41-364, excluding the N-terminal proline-rich sequence. To produce selenomethionine labeled MK2, the protein was expressed in BL21(DE3) (Novagen) E. coli at 25° C. Precultures were grown in shake flasks in LeMaster media supplemented with L-methionine and expression cultures were grown in LeMaster media supplemented with L-selenomethionine that was replenished upon culture induction. Cultures were induced with 0.5 mM IPTG for four hours. Unlabelled MK2 was also expressed in E. coli BL21 (DE3). The culture was induced with 0.5 mM IPTG and cells were harvested 4 hours post-induction.

Purification of MK2 41-364. The purification was performed at 4° C. 5 g of bacterial cells were homogenized in 200 ml of Buffer A (50 mM Tris pH 7.5, 10 mM DTT, 0.24 mg/ml AEBSF)+90 μg/ml TPCK, 2.5 mM Aminobenzamidine, 500 μL protease inhibitor cocktail (without EDTA) for use in purification of poly-(Histidine) tagged proteins (Sigma), RNase, DNase. Cells were lysed by four passages through a Microfluidics microfluidizer submerged in ice. The lysate was collected and centrifuged at 20,000×g for 30 min. The supernatant was applied to a Poros HQ column (Applied Biosystems) that was equilibrated in Buffer A. The flow through was loaded onto a Poros HS column (Applied Biosystems) and the bound protein was eluted with a gradient of Buffer A+1M NaCl. Ammonium sulfate to 0.8 M was added to the peak fraction and the protein was loaded onto a polypropyl aspartamide column (Nest Group) equilibrated with Buffer B (50 mM Hepes pH 7.5, 10 mM DTT, 0.8 M ammonium sulfate). The protein was eluted with a gradient and the peak fraction was concentrated in a Millipore Ultrafree concentrator. The protein was applied to a Superdex 200 column (Amersham Pharmacia Biotech) equilibrated with 20 mM Hepes pH 7.5, 200 mM NaCl, 10 mM DTT. Protein purity was >95%.

Kinetic Analysis.

Materials. ATP, ADP, phosphoenolpyruvate (PEP), NADH, and pyruvate kinase/lactate dehydrogenase enzymes were purchased from Sigma Chemical Co. (St. Louis, Mo.). Activated p38 MAP kinase was purchased from Upstate Biotech (Lake Placid, N.Y.). LSP-1 peptide (RTPKLARQASIELPSM) (SEQ ID NO:10) was purchased from AnaSpec Inc. (San Jose, Calif.).

Activation of MK2 Constructs. The MK2 constructs 41-400 and 41-364 were activated by the phosphorylation of the constructs by p38 MAP kinase. The activation was done in 20 mM HEPES (pH 7.5), 10 mM MgCl₂, 2 mM DTT, 0.50 mM ATP, 0.2 mg/ml MK2 and 0.125 μg activated p38. The reaction was incubated at 25° C. for 1-2 hour then placed on ice to be used for kinetic analysis.

Kinase Kinetics. The rate of MK2 kinase was characterized in 20 mM HEPES (pH 7.5), 10 mM MgCl₂, 2 mM DTT and 100 mM NaCl. The kinetics was followed by linking the turnover of ATP is to the turnover of NADH to NAD. This was followed spectrophotometrically at 340 nm. The continuos assay contained 20 units pyruvate kinase, 30 units lactate dehydrogenase, 0.25 mM NADH, 2 mM PEP and 1.6 to 8.0 μg/mL MK2. For determining the apparent K_(m) for ATP, the ATP concentration was varied from 0.005 to 0.25 mM while the peptide was held constant at 0.2 mM for activated MK-2 or 1.0 mM for the constitutively active form of MK-2 (41-364). For determining the apparent K_(m) for LSP-1, the LSP-1 concentration was varied from 0.01 to 0.5 mM while ATP was held constant at 2 mM. The kinetic analysis was carried out in a 96-well plate at 25° C., on a Molecular Devices spectrophotometer.

Substrate Kinetics. Peptide LSP, based on the protein substrate of MK-2 lymphocyte/leukocyte specific protein, was used for the investigation of the kinetic mechanism for the enzyme MK-2. Data was fit to equation 1 for normal Michaelis-Menten kinetics. v=V _(max) [S]/K _(m) +[S]  (1) where [S] is the substrate, V_(max) is the maximum enzyme velocity, K_(m) is the Michaelis constant.

Crystallization. The selenomethionine labeled protein was concentrated to ˜5 mg/mL according to the Bradford method (31) in a solution containing 20 mM HEPES pH 7.5, 200 mM NaCl, 10 mM DTT, and 5 mM MgCl₂. Prior to crystallization, staurosporine (0.375 mM) was added from a DMSO stock. Diffraction quality conical crystals were obtained at 18° C. from 2M ammonium sulfate, 100 mM HEPES pH 7.5, 2% PEG 400. These crystals, which appeared in 7-10 days, belonged to space group P6₃ with cell dimensions of a=b=160.20 Å, c=133.48 Å and contained four molecules of MK2 in the asymmetric unit. Native MK2 was crystallized in the presence of ADP and the protein solution was prepared as above except that 5 mM ADP was added instead of the staurosporine. Diffraction quality native MK2/ADP co-crystals were obtained at 18° C. from 2.0M ammonium sulfate. These bipyramidal crystals belonged to space group F4₁32 with cell dimensions a=b=c=253.05 Å and contained one molecule of MK2 and one molecule of ADP in the asymmetric unit.

MK2 41-364 also was successfully crystallized using the various conditions described in Table 4. Based on these experiments, it was discovered that MK2 could be crystallized over a broad pH range (4.5 to 8.5). It also was discovered that over that pH range, MK2 could crystallize using a variety of buffers (Cacodylate, Tris, Tris-HCL, Acetate, Malonate, Sodium/Potassium Phosphate, Citrate, HEPES, MES). Additionally, salts of the anions (Sulfate, Citrate, Chloride, Acetate, Phosphate, Malonate and Tartrate) were preferred for crystallization. Still further, the amount of salt needed to crystallize MK2 ranged from 0.1 M to 2.4 M, with less salt required in the presence of polyethylene glycol. However, MK2 preferred to crystallize in the presence of high salt concentrations, defined as 0.8 M or higher, as only three conditions (#1, #14 and #16) have an organic (PEG 400) as the precipitant. Finally, it was discovered that PEG-400, in the range of 2 to 30%, could aid in the crystallization of MK2. It is envisioned that any PEG (39) or its equivalent (e.g., PEG MME, MPD) up to a molecular weight of 3350 could be substituted for PEG 400.

Data Collection.

MK2-Staurosporine. MAD data were collected on beamline 5.O.2 at the ALS, Berkley using an ADSC Quantum-4 CCD detector from a single crystal of the hexagonal SeMet-MK2. The crystal was cooled to −180° C. for data collection and in order to minimize the exposure of the crystal to x-rays, the strategy option within MOSFLM (32) was used to determine the settings that gave the most complete MAD data using the shortest total exposure time. The wavelengths used can be found in Table 2. These data were then used as input to the programs Shake and Bake (33) and ShelX (34) for determination of the Selenium atom positions. Heavy atom parameters for each were refined with SHARP (35). In addition to the MAD data, a higher resolution data set was collected at 1.1 Å.

MK2-ADP. Single-wavelength (1.0 Å) data for the MK2/ADP co-crystals were collected on beamline 5.O.1 at the ALS, Berkley using an ADSC Quantum-4 CCD detector. A single crystal, cooled to −180° C., was used to collect the data set. The data were processed using DENZO/Scalepack (HKL Research, Inc., Charlottesville, Va.) and the statistics from refinement are given-in Table II.

Model Building and Refinement.

MK2-Staurosporine. The structure of the MK2 was built into the original 3.1 Å resolution solvent flattened symmetry-averaged MAD map using the X-AUTOFIT features within QUANTA (Molecular Simulations Inc., San Diego, Calif.). The phases were then extended from 3.1 Å to 2.7 Å with symmetry averaging in DM. This model was then used as the initial model for refinement using the program CNX (35) against the 2.7 Å data. Prior to refinement, 5% of the data were randomly selected and designated as a R_(free) test set to monitor the progress of the refinement. Following seven cycles of refining and rebuilding the refinement converged with a model which contained four molecules of MK2, four staurosporine molecules, 43 water molecules, and two sulfate ions at an R_(cryst) of 23.9% (R_(free) 27.4%). The refinement statistics are given in Table 2.

MK2-ADP. The structure of the MK2/ADP complex was solved using molecular replacement. A composite consisting of the overlapped MK2 monomers from the staurosporine structure was utilized as a molecular replacement probe with AMORE (37). The molecular replacement solution was then rebuilt into a 3.2 Å resolution solvent averaged map. After the initial placement of the protein chain into density, the model was rebuilt utilizing omit maps calculated with BUSTER (38) in order to eliminate the bias from the molecular replacement solution. The structure was refined in CNX using methods as described above. Refinement converged after six rebuilding cycles with a R_(cryst) of 25.9% and a R_(free) of 29.2%. The final model consisted of residues 46-152, 159-217, 227-265, 274-345, and the ADP moiety. The refinement statistics are given in Table 2.

2. Results and Discussion

Alignment of homologues. Map Kap kinase 2 (MK2) is an enzyme that belongs to a family of Map kinase activated protein kinases. Human members of this family include MK2, MK3 and MK5 (23, 24). There is also a MK4 from sea urchin (25). These proteins are highly homologous and, in addition, all have shown to be activated by the Map kinase p38, although to date the only well studied enzyme is MK2 (26, 24). MK2 is phosphorylated on T222, S272, and T334 by p38 and has a putative autophosphorylation site at T338. All four of the phosphorylation sites are conserved in MK3 but only T222 is present in MK5. All of the isozymes have the ATP binding site motif GXGXXG (SEQ ID NO:6), (residues 71-76 in MK2), but only MK2 has the bipartite nuclear localization signal KKIEDDASNPLLLKRRKK (SEQ ID NO:7) (residues 373-389). The putative activation segment is highly conserved in all three isozymes (residues 207-233 in MK2), including the conserved motifs found to flank the activation segments of many kinases, DFG and APE, with the latter being APQ in MK5 (27). Interestingly, the conserved p38 phosphorylation site, T222, is in the activation loop. MK2 and MK3 contain an N-terminal proline rich domain that is absent in MK5 and the C-terminal extension thought to contain the autoinhibitory domain in MK2 is elongated in MK5. Overall, MK2 shares 75% identity with MK3 and 42% identity with MK5.

Analysis of protein constructs. MK2 is a 400 amino acid protein consisting of five domains, an N-terminal proline rich domain, a kinase catalytic domain, a C-terminal kinase autoinhibitory domain, a nuclear export signal, and a nuclear localization sequence, which also the postulated site for p38 binding (28). After analyzing a large number of constructs for expression of MK2 in E. coli, it was found that deleting the first 40 residues of the proline rich domain greatly enhanced the expression levels and solubility of the enzyme. MK2 41-364, a constitutively active form of the enzyme in which a portion of the C-terminal autoinhibitory domain was removed, was purified and produced crystals that diffracted to 2.7 Å. As shown in Table 1, the K_(m) for ATP is very similar for MK2 41-364 as compared to p38 activated MK2 41-400, 7 μM versus 15 μM, respectively. However, binding of the peptide substrate, leukocyte specific protein 1, to MK2 41-364 is more than 40-fold weaker than to the p38 activated MK2 41-400, 584 μM vs. 13 μM K_(m). In addition, the V_(max) for MK2 41-364 is also much lower when compared to the p38 activated MK2 41-400 (0.076 vs. 8.9 μmol/min/mg, respectively). These data indicate that MK2 41-364, which lacks a portion of the autoinhibitory domain, binds ATP normally but peptide substrate binding has been affected dramatically. However, when MK2 41-364 is phosphorylated by p38, the V_(max) of the enzyme is greatly increased to 11 μmol/min/mg, which is comparable to the V_(max) of p38 activated MK2 41-400, 8.9 μmol/min/mg. Phosphorylation of MK2 41-364 also alters the K_(m) for peptide substrate such that the K_(m) decreases from 584 μM to 20 μM, similar to the K_(m) observed in activated MK2 41-400. These data suggest that for optimal substrate binding and activity MK2 must be phosphorylated. Clearly, phosphorylation of key residues within the autoinhibitory C-terminal α helix and T222 in the activation segment induce conformational changes in the enzyme that allow highly efficient binding of peptide substrate (22). Analysis of the MK2 structures in conjunction with other known Ser/THR kinase structures suggests how this may occur.

Analysis of MK2 structures. The MK2 kinase core domain contains an overall fold that is very similar to the structures of other protein kinases. It is bilobal, consisting of a smaller N-terminal domain that is largely β sheet and a larger C-terminal domain dominated by α helices. MK2 shares two structural features within the N-terminal lobe that have been shown to be important in the regulation of many protein kinases (29). These structural elements are the highly conserved αC helix (residues 99-113), and the β1-β2 loop (residues 13-32), that has been termed the phosphate binding loop or “P loop”. The P loop contains the highly conserved ATP binding motif, GXGXXG (SEQ ID NO:6).

The C-terminal lobe contains another important regulatory feature, the MK2 activation segment (residues 207-233), which extends outward from the surface of the catalytic domain into solvent. Electron density was not observed for part of the segment, residues 216-226 including T222, which is one of the p38 phosphorylation sites. The activation segment is likely to be dynamic due to the role the loop is expected to play in the regulation of the enzyme. As discussed previously, MK2 41-364 is a constitutively active truncated version of the enzyme and lacks part of the C-terminal autoinhibitory sequence. Of the four MK2 structures in the asymmetric unit, C-terminal residues 345-364 are disordered in three and residues 358-364 are disordered in the fourth structure. The additional residues ordered in the latter structure are in contact with a symmetry related molecule.

Comparison of the MK2 binary ADP and staurosporine complex structures reveals a conformation change in the P loop where the loop shifts inward to bind to staurosporine and shifts outward to accommodate ADP.

Comparison with other kinases. Although the overall structure of the catalytic domain is highly conserved in many protein kinases, the mechanisms of regulation are quite diverse and in some cases, require dramatic conformational changes (29). The N-terminal and C-terminal lobes of many protein kinases are connected by a flexible hinge (residues 142-145, DGGE (SEQ ID NO:9) in MK2) thus the relative positions of the domains determine whether the kinase is in the “open” or inactive state versus the “closed” or active conformation (29). MK2 can be classified as an “RD” kinase as the catalytic aspartate, D186, is immediately preceded by an arginine, R185. Activation of a number of RD kinases requires phosphorylation on one or more threonine, serine, or tyrosine residues within the activation segment. Activation of MK2 requires phosphorylation, by p38, of the activation segment residue T222. Three protein kinases closely related to MK2, both by structure and shared regulatory mechanisms, are cAMP dependent protein kinase (PKA) (25), Ca⁺⁺/calmodulin dependent protein kinase (cAMK) (19) and Titin (21). All are Ser/Thr kinases and all require phosphorylation of a residue within the activation segment for activation of the kinase. Like MK2, all share an additional level of negative regulation by either a C-terminal autoinhibitory domain or a regulatory subunit. Although PKA lacks an autoinhibitory domain, a bound regulatory subunit maintains the kinase in an inactive conformation. Activation of PKA requires binding of an allosteric regulator, cAMP, to the regulatory subunit resulting in a conformation change and release of the catalytically active kinase subunit. cAMK is maintained in an inactive conformation by a C-terminal autoinhibitory domain that blocks both substrate and ATP binding. Activation of cAMK requires Ca⁺⁺/calmodulin binding which is thought to induce a conformation change resulting in the displacement of the autoinhibitory domain from the peptide and ATP binding sites. Titin also contains a C-terminal autoinhibitory domain that sterically blocks both substrate and ATP binding. Titin activation also requires Ca⁺⁺/calmodulin binding which is also thought to induce a conformation change resulting in the displacement of the autoinhibitory domain from the peptide and ATP binding sites.

The recently published structure of the autoinhibited, inactive form of MK2 47-400 reveals a unique mechanism of kinase regulation (22). The C-terminal autoinhibitory α helix extends along the entire surface of one face of the C-terminal lobe towards the active site and binds, hypothetically, as a pseudosubstrate. Asp366 acts as a phosphothreonine mimetic by coordinating with the basic residues R185 and K212 within the active site of the enzyme. This “pseudosubstrate” region is thought to be positioned in a manner that would effectively block binding of both protein and peptide substrates. The N-terminal lobe of the kinase domain has significant structural differences when compared to the active MK2 structures presented here. The β-2 strand in the MK2 structures of the present invention is replaced by an α helix which effectively disrupts the 5 strand β sheet observed in the structures of the present invention and many other kinases. Additionally, the αC and αD helices are shorter by 1.5 and 1.0 turns, respectively, in the autoinhibited structure.

Analysis of the MK2 active site. The ATP binding site of protein kinases is located in a deep cleft between the N-terminal and C-terminal lobes of the catalytic domain. The β1 and β2 strands in the N-terminal lobe constitute the P loop, which contains a glycine rich motif, GXGXXG (SEQ ID NO:6), is highly conserved in all protein kinases. The conserved glycines confer two important structural properties to the P loop, lack of side chains, which allow loop backbone amides to interact with ATP phosphates without steric hindrance, and backbone flexibility, which allows the P loop to adopt multiple conformations. Conformational flexibility of the P loop is an important factor in the regulation of many protein kinases. The structure of the MK2 P loop, within the context of a 5 strand β-sheet, and the spatial relationship of the αC2 helix are highly consistent with the structures of many protein kinases and also constitute an important part of the kinase active site.

A number of catalytic residues conserved in all protein kinases are spatially oriented to allow efficient transfer of phosphate from ATP to a protein substrate. The binary complex structure of MK2 41-364 and staurosporine is highly consistent with the ternary complex structure of PKA, PKI inhibitor peptide and staurosporine (30) thus forms the basis for a comparative analysis of the MK2 and PKA active sites. The analysis revealed that all active site residues discussed below are spatially conserved. Arg-185 and Asp-186 (R165, D166 in PKA) are catalytic loop residues that are invariant in all “RD” kinases. The catalytic aspartate, D186, is thought to act as a base to remove a proton from the protein substrate hydroxyl group. The phosphates of ATP are positioned for hydrolysis by interactions with backbone amide protons in the P loop and by ionic interactions with K93 (K72 in PKA), which is positioned and stabilized properly by E104 (E91 from PKA), a residue from the αC helix.

The MK2 activation segment, residues 207-233, extends outward from the kinase catalytic domain in a conformation partially stabilized by interactions with another symmetry related MK2 C-terminal domain. The activation segment is also disordered in the structure of the autoinhibited form of MK2 (22). The structural data suggest that the activation segment in unphosphorylated MK2 is highly dynamic and solvent accessible.

The MK2 structures of the present invention are of unphosphorylated enzyme. Although unphosphorylated MK2 41-364 is catalytically active and shows normal binding of ATP, the K_(m) for LSP-1 peptide substrate 45-fold higher and the V_(max) is 100-fold lower when compared to p38 phosphorylated MK2 41-400 (See Table 1). MK2 41-364 phosphorylated by p38, however, exhibits virtually identical peptide substrate binding and catalytic rates as compared to phosphorylated MK2 41-400. These data suggest that p38 phosphorylation of the activation segment residue T222 shifts the equilibrium of the activation segment from an unbound highly dynamic state, as observed in the MK2 structures of the present invention and in the inactive MK2 (22), to a more stable bound state required for efficient binding of peptide substrate and a high catalytic rate. Constitutively active MK2 41-364, however, lacks key residues within the pseudosubstrate region and as observed in the structures of the present invention, the majority of the autoinhibitory C-terminal helix is disordered and probably highly mobile thus allowing a lower level of peptide substrate binding and catalysis. Phosphorylation of MK2 41-364 by p38 restores full enzymatic activity presumably by shifting the equilibrium of the phosphorylated activation segment to the bound state allowing efficient binding of peptide substrate. These data also clearly show that residues 365-400 are not required for full catalytic activity in vitro.

As observed in many other RD kinases, including PKA, that require phosphorylation of a threonine residue within the activation segment for activation and efficient catalysis, two basic residues, R165 and K189 in PKA, are required for coordination and charge neutralization of the phosphoryl threonine. These residues are also spatially conserved in the MK2 structures (R185 and K212).

Differences in ADP and staurosporine structures. Comparison of the MK2 structures co-crystallized with ADP and staurosporine show that the only significant differences lie in the β-sheet containing the GKGING (SEQ ID NO:8) (71-76) loop. This loop, between β strand 1 and 2, and the loop on the other side of β strand 2 that connects to β strand 3 (residues 83-88) move extensively. Ile74 moves 4.7 A closer to Gly209 in the activation loop when bound with staurosporine as compared to ADP where the loop is shifter outward to accommodate ADP. Conversely in the structure with ADP Thr86, found in the loop between β strand 2 and 3, moves closer to the protein core by 2.5 Å as compared to the loop in the structure with staurosporine. This coupled movement of the loops connecting strands 1, 2 and 3 allows the structural integrity of the b-sheet to remain while moving to accommodate different entities in the active site. Excluding the residues in the glycine rich loop, Asp207 and Lys93 make the only significant movement of residues in the active site. In the structure with ADP, these residues adopt orientations in close proximity to the ADP moiety thereby revealing their importance in the catalytic mechanism (29).

Biological implications. Cells exposed to heat shock, cytokines (TNF, IL-1β) or ultraviolet light display an increase in p38 MAP kinase activity due to phosphorylation by the upstream kinases MKK3 and MKK6. p38 in turn phosphorylates a variety of substrates including the transcription factors ATF-2 and CHOP-1, and other kinases such as MK2 and MK3. The p38/MK2 signal transduction cascade plays a pivotal role in the production of proinflammatory cytokines. Mice homozygously deficient in MK2 show a reduction in TNF, IL1β, IL-6 and IFN-γ synthesis and an increased rate of survival upon exposure to LPS, as compared to wild-type mice. MK2 controls the synthesis of cytokines by regulating the translation and/or stability of the encoding mRNAs through the AU-rich elements of the 3′-untranslated regions of the gene. These data indicate that MK2 is a vitally important enzyme in inflammatory based diseases and is a target for anti-inflammatory drug design.

The present invention is based on the determination of a catalytically active MK2 41-364 in complex with ADP and staurosporine. From the structures of the present invention, as well as the previous structure of the inactive enzyme 47-400 (22), it is observed that MK2 is regulated quite differently from other kinases that have a similar fold. Specifically, the autoinhibitory domain of MK2 does not block the nucleotide binding site and that phosphorylation of the residues in the activation domain are necessary for optimal binding of the substrate and activity of the enzyme. The structures of the present invention will permit the design of inhibitors to MK2. TABLE 1 Apparent K_(m) Apparent K_(m) V_(max) Construct p38 (ATP) (μM) (LSP-1) (μM) μmole/min/mg 41-400 − not determined not determined no activity 41-400 + 7.0 + 0.7 13 + 1 8.9 + 0.1 41-364 −  15 + 1.5 584 + 50 0.076 + 0.004 41-364 + not determined 20 + 2 11 + 14 Conditions: p38 (1.25 μg/ml), MK-2 (200 μg/mL), ATP (500 μM), incubate 1-2 hours at 25° C.

TABLE 2 Statistics for data collection and phase determination MK2-Staurosporine MK2-Staurosporine MK2-ADP Remote Peak Inflection SeMet Native Wavelength (Å) λ3 = 0.95666 λ1 = 0.97926 λ2 = 0.97955 1.1 1.0 Resolution Range (Å) 50-3.1 50-3.1 50-3.1 50-2.7 25-3.1 R_(merge)* (%)  6.7(49.2)  6.6(37.7)  6.0(37.9)  4.7(48.1) 5.5(63.1) Completeness (%) 99.6(99.4) 100(100) 100(100) 100(100) 99.3(100.0) Total Reflections 255,756 427,587 449,393 215,726 90,852 Unique Reflections 82,015 42,754 42,927 52,352 12,997 I/σ(I) 10.8(1.7)  16.2(3.4)  18.0(3.6)  20.2(1.6)  34.9(3.3)  F′ (e−)† −2.95 −4.60 −7.21 F″ (e−) 3.70 6.44 1.81 MAD phasing Statistics for MK2-Staurosporine Resolution limits (Å) 6.92 5.24 4.62 4.00 3.70 3.46 3.27 3.10 Overall Phasing power‡ λ 3 anomalous 3.36 2.08 1.72 1.07 0.82 0.68 0.55 0.52 1.08 λ 1 isomorphous 0 0 0 0 0 0 0 0 0 λ 1 anomalous 5.48 4.36 3.58 2.57 1.99 1.64 1.34 1.12 2.39 λ 2 isomorphous 2.72 2.35 1.96 1.59 1.35 1.14 0.93 0.80 1.57 λ 2 anomalous 2.99 1.67 1.33 0.83 0.62 0.53 0.40 0.36 0.80 Mean FOM^(§) 0.85 0.75 0.70 0.60 0.55 0.48 0.42 0.39 0.59 *R_(merge) = Σ|I_(h) − <I_(h)>|/ΣI_(h) where <I_(h)> is the average intensity over symmetry equivalents. Numbers in parentheses reflect statistics for the last shell. †F′ and F″ reported values were refined by SHARP. ‡Phasing power = Σ|F_(h)|/Σ|||F_(PHobs)| − |F_(PHcalc)||, where F_(h) is the calculated heavy atom structure-factor amplitude. ^(§)Figure of Merit = <ΣP(a)e^(ia)/Σ|P(α)|>, where α is the phase and P(α) is the phase probability distribution.

TABLE 3 Residues of MK2 within 4 Å of staurosporin in MK2/staurosporine complex Leu70, Gly71, Leu72, Gly73, Val78, Ala91, Val118, Mse138, Glu139, Cys140, Leu141, Glu145, Glu190, Asn191, Leu192, Thr206, Asp207 Residues of MK2 within 8 Å of staurosporin in MK2/staurosporine complex Val69, Leu70, Gly71, Leu72, Gly73, Ile74, Gly76, Ala77, Val78, Leu79, Gln80, Lys89, Phe90, Ala91, Leu92, Lys93, Leu95, Glu104, His108, Val118, Arg119, Ile136, Val137, Mse138, Glu139, Cys140, Leu141, Asp142, Gly143, Gly144, Glu145, His184, Asp186, Lys188, Pro189, Glu190, Asn191, Leu192, Leu193, Tyr194, Thr195, Lys204, Leu205, Thr206, Asp207, Phe208, Gly209 Residues of MK2 within 4 Å of ADP in MK2/ADP complex Leu70, Gly71, Leu72, Gly73, Ile74, Asn75, Val78, Ala91, Lys93, Met138, Glu139, Cys140, Leu141, Asn191, Thr206, Asp207 Residues of MK2 within 8 Å of ADP in MK2/ADP complex Val69, Leu70, Gly71, Leu72, Gly73, Ile74, Asn75, Gly76, Ala77, Val78, Leu79, Gln80, Phe90, Ala91, Leu92, Lys93, Met94, Leu95, Glu104, His108, Val118, Ala119, Ile136, Val137, Met138, Glu139, Cys140, Leu141, Asp142, Gly143, Gly144, Glu145, His184, Asp186, Lys188, Pro189, Glu191, Asn191, Leu192, Leu193, Tyr194, Thr195, Leu205, Thr206, Asp207, Phe208, Gly209, Phe210

TABLE 4 Precipitant Buffer Additional Salt #1 30% (v/v) 0.1M Cacodylate pH 6.5 0.2M Lithium Sulfate polyethyleneglycol-400 #2 1.0 M sodium citrate 0.1M Tris pH 7.0 0.2M Sodium chloride #3 0.8 M sodium dihydrogen 0.2 M Acetate pH 4.5 phosphate 1.2M K₂HPO₄ dipotassium hydrogen phosphate #4 1.0M sodium citrate 0.1M Cacodylate pH 6.5 #5 1.9 M sodium malonate pH 5.0 #6 1.5 M sodium malonate pH 6.0 #7 1.5 M sodium malonate pH 7.0 #8 2.0 M ammonium Sulfate #9 1/17M NaH₂PO₄ 0.63M K₂HPO₄ (Final: 1.8M Sodium/ Potassium Phosphate pH 6.3) #10 0.27M NaH₂PO₄ 1.53M K₂HPO₄ (Final: 1.8M Sodium/ Pottassium Phosphate pH 7.5) #11 0.072M NaH₂PO₄ 1.728M K₂HPO₄ (Final: 1.8M Sodium/ Pottassium Phosphate pH 8.2) #12 1.6M ammonium sulfate 0.1M citric acid pH 5.0 #13 2.4M ammonium sulfate 0.1M HEPES pH 7.0 #14 30% v/v polyethyleneglycol- 0.1M Tris hydrochloride pH 8.5 0.2M tri-Sodium citrate 400 dihydrate #15 1M potassium sodium 0.1M HEPES-Na pH 7.5 tartrate tetrahydrate #16 18% v/v polyethyleneglycol- 0.1M HEPES-Na pH 7.5 0.1M ammonium sulfate 400 #17 1M tri-sodium citrate 0.1M HEPES-Na pH 7.5 dihydrate #18 1.6M Sodium/Potassium 0.1M MES pH 6.5 Tartrate #19 2.0M Ammonium Sulfate 0.1M HEPES pH 7.5 2% PEG 400

REFERENCES

1. Lee, J. C., Laydon, J. T., McDonnel, P. C., Gallagher, T. F., Kumar, S., Green, D., McNulty, D., Blumenthal, M. J., Heys, J. R., Landvatter, S. W., al., e. (1994) A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372, 739-746.

2. Cuenda, A., Rouse, J., Doza, Y. N., Meier, R., Cohen, P., Gallagher, T. F., Young, P. R., Lee, J. C. (1995) SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Letters 364, 229-233.

3. Kotlyarov, A., Neininger, A., Schubert, C., Eckert, R., Birchmeier, C., Volk, H.-D., Gaestel, M. (1999) MAPKAP kinase 2 is essential for LPS-induced TNF-biosynthesis. Nat. Cell. Biol. 1, 94-97.

4. Neininger, A., Kontoyiannis, D., Kotlyarov, A., Winzen, R., Eckert, R., Volk, H.-D., Holtmann, H., Kollias, G., Gaestel, M. (2002) MK2 Targets AU-rich Elements and Regulates Biosynthesis of Tumor Necrosis Factor and Interleukin-6 Independently at Different Post-transcriptional Levels. J. Biol. Chem. 277, 3065-3068.

5. Tan, Y., Rouse, J., Zhang, A., Cariati, S., Cohen, P., Comb, M. (1996) FGF and stress regulate CREB and ATF-1 via a pathway involving p38 MAP kinase and MAPKAP kinase-2. EMBO J. 15, 4629-4642.

6. Lavoie, J., Hickey, E., Weber, L., Landry, J. (1993) Modulation of actin microfilament dynamics and fluid phase pinocytosis by phosphorylation of heat shock protein 27. J. Biol. Chem. 268, 24210-24214.

7. Stokoe, D., Engel, K., Campbell, D. G., Cohen, P., Gaestel, M. (1992) Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Letters 313, 307-313.

8. Ben-Levy, R., Leighton, I., Doza, Y., Attwood, P., Morrice, N., Marshall, C., Cohen, P. (1995) Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2. EMBO J. 14, 5920-5930.

9. Hedges, J. C., Dechert, M. A., Yamboliev, I. A., Martin, J. L., Hickey, E., Weber, L. A., Gerthoffer, W. T. (1999) A Role for p38MAPK/HSP27 Pathway in Smooth Muscle Cell Migration. J. Biol. Chem. 274, 24211-24219.

10. Kotlyarov, A., Yannoni, Y., Fritz, S., Laass, K., Telliez, J.-B., Pitman, D., Lin, L.-L., Gaestel, M. (2002) Distinct Cellular Functions of MK2. Mol. Cell. Biol. 22, 4827-4835.

11. Stokoe, D., Caudwell, B., Cohen, P. T., Cohen, P. (1993) The substrate specificity and structure of mitogen-activated protein (MAP) kinase-activated protein kinase-2. Biochem. J 296, 843-849.

12. Engel, K., Kotlyarov, A., Gaestel, M. (1998) Leptomycin B-sensitive nuclear export of MAPKAP kinase 2 is regulated by phosphorylation. EMBO J. 17, 3363-3371.

13. Ben-Levy, R., Hooper, S., Wilson, R., Paterson, H. F., Marshall, C. J. (1998) Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2. Curr. Biol. 8, 1049-1057.

14. Engel, K., Plath, K., Gaestel, M. (1993) The MAP kinase-activated protein kinase 2 contains a proline-rich SH3-binding domain. FEBS Letters 336, 143-147.

15. Veron, M., Radzio-Andzelm, E., Tsigelny, I., Eyck, L., Taylor, S. (1993) A Conserved Helix Motif Complements the Protein Kinase Core. Proc. Natl. Acad. Sci. 90, 10618-10622.

16. Zu, Y.-L., Wu, F., Gilchrist, A., Ai, Y., Labadia, M. E., Huang, C.-K. (1994) The Primary Structure of a Human MAP Kinase Activated Protein Kinase 2. Biochem. Biophys. Res. Commun. 200, 1118-1124.

17. Stokoe, D., Campbell, D., Nakielny, S., Hidaka, H., Leevers, S., Marshall, C., Cohen, P. (1992) MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase. EMBO J. 11, 3985-3994.

18. Engel, K., Schultz, H., Martin, F., Kotlyarov, A., Plath, K., Hahn, M., Heinemann, U., Gaestel, M. (1995) Constitutive Activation of Mitogen-activated Protein Kinase-activated Protein Kinase 2 by Mutation of Phosphorylation Sites and an A-helix Motif. J. Biol. Chem. 270, 27213-27221.

19. Zheng, J. H., Trafny, E. A., Knighton, D. R., Xuong, N. H., Taylor, S. S., Teneyck, L. F., Sowadski, J. M. (1993) Crystal structure of the catalytic subunit of cAMP-dependent protein kinase complexed with magnesium-ATP and peptide inhibitor. Biochemistry 32, 2154-61.

20. Goldberg, J., Nairn, A. C., Kuriyan, J. (1996) Structural basis for the autoinhibition of calcium/calmodulin-dependent protein kinase I. Cell 84, 875-87.

21. Mayans, O., van der Ven, P. F. M., Wilm, M., Mues, A., Young, P., Furst, D. O., Wilmanns, M., Gautel, M. (1998) Structural basis for activation of the titin kinase doamin during myofibrillogenesis. Nature 395, 863-869.

22. Meng, W., Swenson, L. L., Fitzgibbon, M. J., Hayakawa, K., Haar, E. t., Behrens, A. E., Fulghum, J. R., Lippke, J. A. (2002) Structure of MAPKAP kinase 2 suggests a bifunctional switch that couples kinase ativation with nuclear export. J. Biol. Chem., 37401-37405.

23. McLaughlin, M. M., Kumar, S., McDonnell, P. C., Van Horn, S., Lee, J. C., Livi, G. P., Young, P. R. (1996) Identification of Mitogen-activated Protein (MAP) Kinase-activated Protein Kinase-3, a Novel Substrate of CSBP p38 MAP Kinase. J. Biol. Chem. 271, 8488-8492.

24. Ni, H., Wang, X. S., Diener, K., Yao, Z. (1998) MAPKAPK5, a Novel Mitogen-Activated Protein Kinase (MAPK)-Activated Protein Kinase, Is a Substrate of the Extracellular-Regulated Kinase (ERK) and p38 Kinase. Biochem. Biophys. Res. Commun. 243, 492-496.

25. Komatsu, S., Murai, N., Totsukawa, G., Abe, M., Akasaka, K., Shimada, H., Hosoya, H. (1997) Identification of MAPKAPK homolog (MAPKAPK-4) as a myosin II regulatory light-chain kinase in sea urchin egg extracts. Arch. Biochem. Biophys. 343, 55-62.

26. Clifton, A. D., Young, P. R., Cohen, P. (1996) A comparison of the substrate specificity of MAPKAP kinase-2 and MAPKAP kinase-3 and their activation by cytokines and cellular stress. FEBS Letters 392, 209-214.

27. Johnson, L. N., Noble, M. E. M., Owen, D. J. (1996) Active and Inactive Protein Kinases: Structural Basis for Regulation. Cell 85, 149-158.

28. Tanoue, T., Adachi, M. Moriguchi, T., Nishida, E. (2000) A conserved docking motif in MAP kinases common to substrates, activators and regulators. Nat. Cell. Biol. 2, 110-116.

29. Huse, M., Kuriyan, J. (2002) The Conformational Plasticity of Protein Kinases. Cell 109, 275-282.

30. Prade, L., Engh, R. A., Girod, A., Kinzel, V., Huber, R., Bossemeyer, D. (1997) Staurosporine-induced conformational changes of cAMP-dependent protein kinase catalytic subunit explain inhibitory potential. Structure 5, 1627-37.

31. Bradford, M. M. (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry 72, 248-254.

32. Leslie, A. G. W. (1992) Recent changes to the MOSFLM package for processing film and image plate data. Joint CCP4+ESF-EAMCB Newsletter on Protein Crystallography 26.

33. Weeks, C. M., Miller, R. (1999) The design and implementation of SnB v2.0. J. App. Crystallogr. 32, 120-124.

34. Sheldrick, G. M. (1997) Patterson Superposition and ab Initio Phasing. Methods in Enzymol. 276, 628-641.

35. De La Fortelle, E., Bricogne, G. (1997) Maximum-likelihood heavy atom parameter refinement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472-494.

36. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S. (1998) Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D54, 905-921.

37. CCP4 (1994) The CCP-4 suite: programs for X-ray crystallography. Acta Crystallogr. D50, 760-763.

38. Bricogne, G. (1993) Direct phase determination by entropy maximization and likelihood ranking; status report and perspectives. Acta Crystallogr. D54, 905-921.

39. McPherson, A. (1999) Crystallization of Biological Macromolecules, Cold Spring Harbor Press, Cold Spring Harbor.

All publications mentioned herein above, whether to issued patents, pending applications, published articles, or otherwise, are hereby incorporated by reference in their entirety. While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of the disclosure that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims. 

1-10. (canceled)
 11. A method for obtaining crystallized native MK2, crystallized MK2 polypeptide or crystallized MK2 analogue comprising contacting native MK2, MK2 polypeptide or MK2 analogue with a buffer solution comprising at least one of cacodylate, Tris, Tris-HCL, acetate, malonate, sodium phosphate, potassium phosphate, citrate, HEPES and MES, at a salt concentration of 0.1 M to 2.4 M, and at a pH of 4.5 to 8.5, under conditions permitting the formation of crystallized MK2, crystallized MK2 polypeptide or crystallized MK2 analogue.
 12. The method of claim 11, wherein the salt has an anion selected from the group consisting of sulfate, citrate, chloride, acetate, phosphate, malonate and tartrate.
 13. The method of claim 11, wherein the salt concentration is 0.8 M or higher.
 14. The method of claim 11, wherein native MK2, MK2 polypeptide or MK2 analogue is contacted with the buffer solution in the presence of PEG or a PEG substitute having a molecular weight up to
 3350. 15. The method of claim 14, wherein PEG or PEG substitute is selected from the group consisting of PEG-200, PEG-400, PEG-500-MME, PEG-1000, PEG-1500, PEG-2000-MME, PEG-3350, Jeffamine M-600, ethylene glycol, glycerol and 1-6 hexanediol, 2-methyl-2,4-pentanediol (MPD).
 16. The method of claim 14, wherein PEG is PEG-400.
 17. A crystallized complex comprising an MK2 polypeptide and staurosporine.
 18. The crystallized complex of claim 17, characterized as having space group P6₃, unit cell parameters of a=b=160.20 Å, c=133.48 Å.
 19. A crystallized complex comprising an MK2 polypeptide and ADP.
 20. The crystallized complex of claim 19, characterized as having space group F4₁32, unit cell parameters of a=b=c=253.05 Å. 21-57. (canceled)
 58. The crystallized complex of claim 17, wherein the complex comprises four molecules of MK2 polypeptide per asymmetric unit.
 59. The crystallized complex of claim 17, wherein the complex further comprises selenomethionine.
 60. The crystallized complex of claim 17, wherein the complex diffracts to at least 3.1 Å resolution.
 61. The crystallized complex of claim 17, wherein the complex diffracts to at least 1.1 Å resolution.
 62. The crystallized complex of claim 19, wherein the complex comprises one molecule of MK2 polypeptide per asymmetric unit.
 63. The crystallized complex of claim 19, wherein the complex diffracts to at least 1.0 Å resolution.
 64. The crystallized complex of claim 17, wherein the MK2 polypeptide comprises amino acid residues Leu70, Gly71, Leu72, Gly73, Val78, Ala91, Val118, Mse138, Glu139, Cys140, Leu141, Glu145, Glu190, Asn191, Leu192, Thr206, and Asp207 as set forth in SEQ ID NO:1.
 65. The crystallized complex of claim 64, wherein the MK2 polypeptide further comprises amino acid residues Val69, Ile74, Gly76, Ala77, Leu79, Gln80, Lys89, Phe90, Leu92, Lys93, Leu95, Glu104, His108, Arg 119, Ile136, Val137, Asp142, Gly143, Gly144, His184, Asp186, Lys188, Pro189, Leu193, Tyr194, Thr195, Lys204, Leu205, Phe208, and Gly209 as set forth in SEQ ID NO:
 1. 66. The crystallized complex of claim 17, wherein the crystallized complex has the structural coordinates set forth in FIGS. 2 through 2A-204 ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å.
 67. The crystallized complex of claim 17, wherein the MK2 polypeptide comprises an active site comprising the relative structural coordinates of amino acid residues Leu70, Gly71, Leu72, Gly73, Val78, Ala91, Val118, Mse138, Glu139, Cys140, Leu141, Glu145, Glu190, Asn191, Leu192, Thr206, and Asp207 of molecules A, B, C or D according to the sequence set forth in SEQ ID NO:1, and the structural coordinates as set forth in FIGS. 2 through 2A-204 ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å.
 68. The crystallized complex of claim 67, wherein the active site of the MK2 polypeptide further comprises the relative structural coordinates of amino acid residues Val69, Ile74, Gly76, Ala77, Leu79, Gln80, Lys89, Phe90, Leu92, Lys93, Leu95, Glu104, His108, Arg119, Ile136, Val137, Asp142, Gly143, Gly144, His184, Asp186, Lys188, Pro189, Leu193, Tyr194, Thr195, Lys204, Leu205, Phe208, and Gly209 of molecules A, B, C or D according to the sequence set forth in SEQ ID NO:1, and the structural coordinates as set forth in FIGS. 2 through 2A-204 ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å.
 69. The crystallized complex of claim 19, wherein the MK2 polypeptide comprises amino acid residues Leu70, Gly71, Leu72, Gly73, Val78, Ala91, Val118, Mse138, Glu139, Cys140, Leu141, Glu145, Glu190, Asn191, Leu192, Thr206, and Asp207 as set forth in SEQ ID NO:1.
 70. The crystallized complex of claim 69, wherein the MK2 polypeptide further comprises amino acid residues Val69, Ile74, Gly76, Ala77, Leu79, Gln80, Lys89, Phe90, Leu92, Lys93, Leu95, Glu104, His108, Arg119, Ile136, Val237, Asp142, Gly143, Gly144, His184, Asp186, Lys188, Pro189, Leu193, Tyr194, Thr195, Lys204, Leu205, Phe208, and Gly209 as set forth in SEQ ID NO:1.
 71. The crystallized complex of claim 19, wherein the crystallized complex has the structural coordinates set forth in FIGS. 3 through 3A-40 ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å.
 72. The crystallized complex of claim 19, wherein the MK2 polypeptide comprises an active site comprising the relative structural coordinates of amino acid residues Leu70, Gly71, Leu72, Gly73, Val78, Ala91, Val118, Mse138, Glu139, Cys140, Leu141, Glu145, Glu190, Asn191, Leu192, Thr206, and Asp207 of molecule A according to the sequence set forth in SEQ ID NO:1, and the structural coordinates as set forth in FIGS. 3 through 3A-40 ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å.
 73. The crystallized complex of claim 72, wherein the active site of the MK2 polypeptide further comprises the relative structural coordinates of amino acid residues Val69, Ile74, Gly76, Ala77, Leu79, Gln80, Lys89, Phe90, Leu92, Lys93, Leu95, Glu104, His108, Arg119, Ile136, Val137, Asp142, Gly143, Gly144, His184, Asp186, Lys188, Pro189, Leu193, Tyr194, Thr195, Lys204, Leu205, Phe208, and Gly209 of molecule A according to the s set forth in SEQ ID NO:1, and the structural coordinates as set forth in FIGS. 3 through 3A-40 ±a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å. 